Fluorescence in situ hybridization (FISH) has proven to be a valuable tool for cytogenetic research. The procedure reveals the chromosomal location of DNA target sequences with homology to labeled nucleic acid probes. As an example, inversions in DNA sequences are known to be associated with tumors and birth defects. Since hybridization is dependent on nucleotide pairing through hydrogen bonding, it requires that both probe and target be functionally single stranded initially. Probes may be either single stranded at the time of their construction or made single stranded by denaturation with heat or a high or low pH. When the target is double stranded, such as the DNA of chromosomes, denaturation is also required prior to hybridization. With FISH, this is most commonly done by thermal denaturation. Enzymatic digestion with exonuclease III (Exo III) has also been used to obtain a single-stranded target.
Therefore, preparation of single-stranded chromatids for determination of chromosomal orientation and strand direction of DNA sequences is receiving increased attention. Conventional fluorescence in situ hybridization methods rely on nucleotide base pairing between a labeled probe and the complementary chromosomal target sequence. Both probe and target DNAs must be functionally single-stranded at the start of the procedure. Single-stranded probes can be constructed or prepared, but the chromosomal DNA must be made single-stranded by denaturation. Because both strands are present in both chromatids of each chromosome, even single-stranded probes will hybridize to both chromatids of the target chromosome.
Selectively removing one strand of the DNA double helix from each chromatid makes strand-specific fluorescence in situ hybridization (FISH) possible. Single-stranded probes can then be hybridized to single-stranded chromosomal DNA without denaturation. Probes of repetitive sequences arranged head-to-tail in tandem arrays will hybridize only to one chromatid. By contrast, probes of ubiquitous repeat sequences, such as Alu, that are present in both directions on each DNA strand, will hybridize to both chromatids. Thus, the method reveals the orientation of repetitive sequences and has been designated as CO-FISH for chromosome orientation-FISH. This process is schematically illustrated in FIGS. 1a-e hereof, labeled Prior Art, and is reported in "Strand-Specific FISH Reveals Orientation Of Chromosome 18 Alphoid DNA," by E. H. Goodwin and J. Meyne, Cytogenet. Cell Genet., 63, 126 (1993), the teachings of which are hereby incorporated by reference herein.
In its broadest embodiment, the manner in which FISH can be made strand specific of Goodwin et al., supra, may be understood as follows. First, cell cultures are grown in a medium containing a halogenated nucleotide analogue such as bromodeoxyuridine (BrdU) (FIG. 1a). Due to the semiconservative nature of DNA synthesis, the two newly replicated chromatids are singly substituted; that is, the nucleotide analogue is partially substituted for thymidine in one DNA strand of each chromatid (FIG. 1b). Nucleotide analogue incorporation takes place in opposite strands of the two sister chromatids; each replicated chromosome now containing sister chromatids that are singly substituted in opposite DNA strands. Metaphase chromosome spreads are prepared by standard methods. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand breaks that are thought to occur preferentially at nucleotide analogue incorporation sites. Nicks produced in chromosomal DNA by this treatment are substrates for enzymatic digestion by Exo III which excises nucleotides from one strand of double-stranded DNA starting at the sites of the nicks. In theory, the process should remove most of the nucleotide analogue incorporated strand while leaving the original (prereplication) strand largely intact (FIG. 1c). The single strands remaining in each of the two chromatids are complementary. Thus, chromatids can not only be made single stranded, but the sister chromatids contain only complementary DNA strands. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present (FIG. 1d). Finally, detection of the hybridization site will demonstrate fluorescence on only one chromatid (FIG. 1e).
Slides prepared for the CO-FISH method can also be used to determine the direction of the DNA strand to which the single-stranded probe hybridizes. This procedure is referred to as chromosome orientation and direction FISH, or COD-FISH, and is schematically illustrated in FIG. 2a,b hereof, labeled Prior Art. The C-rich telomere probe is co-hybridized with a single-stranded probe of the sequence of interest (FIG. 2a). Because the G-rich telomere sequence is located at the 3' end of the DNA strand, the C-rich strand probe determines the 3' end of the DNA strands in each chromatid. The sequence of interest will hybridize to its complementary sequence. The 5'-to-3' direction of each strand can be determined by the position of the telomere fluorescence (FIG. 2b). The direction of the sequence of interest can be inferred from this information.
Multiple color and sequential hybridizations can be used on CO-FISH slides. Two or more single-stranded probes with different molecular tags can be hybridized at once, in a similar manner to that for standard FISH methods. Sequential hybridizations are simple. After the first hybridization, the slide is rinsed in 2.times. SSC (0.3 M NaCl+0.03 M sodium citrate) to remove any excess probe from the slide and then drained to remove excess 2.times. SSC, but not allowed to dry. The strand may then be again hybridized with another probe. Sequential hybridizations are preferred for two color hybridizations with complementary strands of repeat probes. Four sequential hybridizations have been achieved by the present inventors on a single slide. It is believed that the only limitation is the background hybridization after detection of the signal. However, this problem may be reduced by using probes labeled directly with fluorescent nucleotides rather than immunofluorescence detection. Essentially any method useful for in situ hybridization is applicable to the CO-FISH method.
Bromodeoxycytidine, the cytidine analogue was observed to produce similar results to BrdU.
Accordingly, it is an object of the present invention to prepare single stranded sister chromatids containing only complementary DNA strands without the use of an exonuclease.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.